Nonlinear Mixed-Effects Model of Z-Endoxifen Concentrations in Tamoxifen-Treated Patients from the CEPAM Cohort.

Tamoxifen is widely used in patients with hormone receptor-positive breast cancer. The polymorphic enzyme CYP2D6 is primarily responsible for metabolic activation of tamoxifen, resulting in substantial interindividual variability of plasma concentrations of its most important metabolite, Z-endoxifen. The Z-endoxifen concentration thresholds below which tamoxifen treatment is less efficacious have been proposed but not validated, and prospective trials of individualized tamoxifen treatment to achieve Z-endoxifen concentration thresholds are considered infeasible. Therefore, we aim to validate the association between Z-endoxifen concentration and tamoxifen treatment outcomes, and identify a Z-endoxifen concentration threshold of tamoxifen efficacy, using pharmacometric modeling and simulation. As a first step, the CYP2D6 Endoxifen Percentage Activity Model (CEPAM) cohort was created by pooling data from 28 clinical studies (> 7,000 patients) with measured endoxifen plasma concentrations. After cleaning, data from 6,083 patients were used to develop a nonlinear mixed-effect (NLME) model for tamoxifen and Z-endoxifen pharmacokinetics that includes a conversion factor to allow inclusion of studies that measured total endoxifen but not Z-endoxifen. The final parent-metabolite NLME model confirmed the primary role of CYP2D6, and contributions from body weight, CYP2C9 phenotype, and co-medication with CYP2D6 inhibitors, on Z-endoxifen pharmacokinetics. Future work will use the model to simulate Z-endoxifen concentrations in patients receiving single agent tamoxifen treatment within large prospective clinical trials with long-term survival to identify the Z-endoxifen concentration threshold below which tamoxifen is less efficacious. Identification of this concentration threshold would allow personalized tamoxifen treatment to improve outcomes in patients with hormone receptor-positive breast cancer.

Protocol for establishing a coculture with fibroblasts and colorectal cancer organoids.

The tumor microenvironment is essential for mediating drug resistance and tumor progression. Here, we present a coculture system, which enables drug testing of colorectal cancer organoids and fibroblasts without additional matrix components such as Matrigel or basement membrane extracts. First, we describe steps to use a readout for high-throughput drug testing using a luminescence-based viability assay. Second, we detail a readout that uses flow cytometry to distinguish toxic effects on either colorectal cancer organoids or fibroblasts.

Identification and synthesis of (Z)-3'-hydroxy clomiphene as a new potential doping-relevant metabolite of clomiphene.

A recent study addressed the possibility of unintentional ingestion of clomiphene through residues in chicken eggs. The method developed here helped distinguish between microdose intake of (E/Z)-clomiphene citrate and consumption of clomiphene-containing eggs by the urinary pattern of four mono-hydroxylated clomiphene metabolites. However, reanalyses of doping-control samples, which showed an adverse analytical finding for clomiphene, revealed a hydroxy clomiphene (HC) isomer that was not found after microdose intake or after consumption of clomiphene-containing eggs and could not be assigned to any of the available reference compounds. The aim of the present follow-up study was to identify this HC isomer and to characterize this metabolite with respect to its potential properties as long-term metabolite in doping controls. METHODS: (E/Z)-3'-HC and (E/Z)-4'-HC were synthesized involving the McMurry reaction. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and optimized after a derivatization step with dansyl chloride to separate eight HC isomers. Using this method, urine samples from a controlled clomiphene administration study were analyzed, in which male study participants received therapeutic doses of clomiphene for 30 days and collected urine samples for up to 8 months. Thus, isomer-specific HC elimination profiles could be monitored. RESULTS: The metabolite previously found in doping-control samples was identified as (Z)-3'-HC. The elimination profiles of the different HCs confirmed previous results, with (Z)-3-HC being the most abundant urinary hydroxy metabolite shortly after administration. A new finding was that the data suggest that (Z)-3'-HC is excreted at higher relative concentrations only several weeks after drug intake. CONCLUSION: These findings might be of particular importance in sport drug testing as they can assist in the decision-making process to distinguish between intentional doping and inadvertent exposure to clomiphene via food contamination.

Performance comparison of narrow-bore and capillary liquid-chromatography for non-targeted metabolomics profiling by HILIC-QTOF-MS.

Clinical metabolomics studies often have to cope with limited sample amounts, thus miniaturized liquid chromatography (LC) systems are a promising alternative. Their applicability has already been demonstrated in various fields, including a few metabolomics studies that mainly used reversed-phase chromatography. However, hydrophilic interaction chromatography (HILIC), which is widely used in metabolomics due to its particular suitability for the analysis of polar molecules, has rarely been tested for miniaturized LC-MS analysis of small molecules. In the present work, the suitability of a capillary HILIC (CapHILIC)-QTOF-MS system for non-targeted metabolomics was evaluated based on extracts of porcine formalin-fixed, paraffin-embedded (FFPE) tissue samples. The performance was assessed with respect to the number and retention time span of metabolic features as well as the analytical repeatability, the signal-to-noise ratio and the signal intensity of 16 annotated metabolites from different compound classes. The results were compared with a well established narrow-bore HILIC-QTOF-MS system. Both platforms have detected a similar number of features and performed excellent with respect to retention time stability (median RT span <0.05 min) and analytical repeatability (>75% of features with CV < 20%). The signal areas of all metabolites assessed were increased up to 18-fold by the use of CapHILIC, although the signal-to-noise ratio was only improved for 50% of the metabolites. An even better reproducibility (median CV = 5.2%) and up to 80-fold increase in signal intensity were observed after optimization of CapHILIC conditions for analysis of bile acid standard solutions. Even though the observed improvement for specific bile acids (e.g. taurocholic acid) in biological matrix needs to be evaluated, the platform comparison indicates, that the tested CapHILIC system is particularly suitable for analyses of a less broad metabolite spectrum, and specifically optimized chromatography.

Perfusion Air Culture of Precision-Cut Tumor Slices: An Ex Vivo System to Evaluate Individual Drug Response under Controlled Culture Conditions.

Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air-liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.

Toward model-informed precision dosing for tamoxifen: A population-pharmacokinetic model with a continuous CYP2D6 activity scale.

BACKGROUND: Tamoxifen is important in the adjuvant treatment of breast cancer. A plasma concentration of the active metabolite endoxifen of > 16 nM is associated with a lower risk of breast cancer-recurrence. Since inter-individual variability is high and > 20 % of patients do not reach endoxifen levels > 16 nM with the standard dose tamoxifen, therapeutic drug monitoring is advised. However, ideally, the correct tamoxifen dose should be known prior to start of therapy. Our aim is to develop a population pharmacokinetic (POP-PK) model incorporating a continuous CYP2D6 activity scale to support model informed precision dosing (MIPD) of tamoxifen to determine the optimal tamoxifen starting dose. METHODS: Data from eight different clinical studies were pooled (539 patients, 3661 samples) and used to develop a POP-PK model. In this model, CYP2D6 activity per allele was estimated on a continuous scale. After inclusion of covariates, the model was subsequently validated using an independent external dataset (378 patients). Thereafter, dosing cut-off values for MIPD were determined. RESULTS: A joint tamoxifen/endoxifen POP-PK model was developed describing the endoxifen formation rate. Using a continuous CYP2D6 activity scale, variability in predicting endoxifen levels was decreased by 37 % compared to using standard CYP2D6 genotype predicted phenotyping. After external validation and determination of dosing cut-off points, MIPD could reduce the proportion of patients with subtherapeutic endoxifen levels at from 22.1 % toward 4.8 %. CONCLUSION: Implementing MIPD from the start of tamoxifen treatment with this POP-PK model can reduce the proportion of patients with subtherapeutic endoxifen levels at steady-state to less than 5 %.

Cross-Ancestry Genome-Wide Association Study Defines the Extended CYP2D6 Locus as the Principal Genetic Determinant of Endoxifen Plasma Concentrations.

The therapeutic efficacy of tamoxifen is predominantly mediated by its active metabolites 4-hydroxy-tamoxifen and endoxifen, whose formation is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6). Yet, known CYP2D6 polymorphisms only partially determine metabolite concentrations in vivo. We performed the first cross-ancestry genome-wide association study with well-characterized patients of European, Middle-Eastern, and Asian descent (n = 497) to identify genetic factors impacting active and parent metabolite formation. Genome-wide significant variants were functionally evaluated in an independent liver cohort (n = 149) and in silico. Metabolite prediction models were validated in two independent European breast cancer cohorts (n = 287, n = 189). Within a single 1-megabase (Mb) region of chromosome 22q13 encompassing the CYP2D6 gene, 589 variants were significantly associated with tamoxifen metabolite concentrations, particularly endoxifen and metabolic ratio (MR) endoxifen/N-desmethyltamoxifen (minimal P = 5.4E-35 and 2.5E-65, respectively). Previously suggested other loci were not confirmed. Functional analyses revealed 66% of associated, mostly intergenic variants to be significantly correlated with hepatic CYP2D6 activity or expression (ρ = 0.35 to -0.52), and six hotspot regions in the extended 22q13 locus impacting gene regulatory function. Machine learning models based on hotspot variants (n = 12) plus CYP2D6 activity score (AS) increased the explained variability (~ 9%) compared with AS alone, explaining up to 49% (median R2 ) and 72% of the variability in endoxifen and MR endoxifen/N-desmethyltamoxifen, respectively. Our findings suggest that the extended CYP2D6 locus at 22q13 is the principal genetic determinant of endoxifen plasma concentration. Long-distance haplotypes connecting CYP2D6 with adjacent regulatory sites and nongenetic factors may account for the unexplained portion of variability.

Prediction of Drug-Drug-Gene Interaction Scenarios of (E)-Clomiphene and Its Metabolites Using Physiologically Based Pharmacokinetic Modeling.

Clomiphene, a selective estrogen receptor modulator (SERM), has been used for the treatment of anovulation for more than 50 years. However, since (E)-clomiphene ((E)-Clom) and its metabolites are eliminated primarily via Cytochrome P450 (CYP) 2D6 and CYP3A4, exposure can be affected by CYP2D6 polymorphisms and concomitant use with CYP inhibitors. Thus, clomiphene therapy may be susceptible to drug-gene interactions (DGIs), drug-drug interactions (DDIs) and drug-drug-gene interactions (DDGIs). Physiologically based pharmacokinetic (PBPK) modeling is a tool to quantify such DGI and DD(G)I scenarios. This study aimed to develop a whole-body PBPK model of (E)-Clom including three important metabolites to describe and predict DGI and DD(G)I effects. Model performance was evaluated both graphically and by calculating quantitative measures. Here, 90% of predicted Cmax and 80% of AUClast values were within two-fold of the corresponding observed value for DGIs and DD(G)Is with clarithromycin and paroxetine. The model also revealed quantitative contributions of different CYP enzymes to the involved metabolic pathways of (E)-Clom and its metabolites. The developed PBPK model can be employed to assess the exposure of (E)-Clom and its active metabolites in as-yet unexplored DD(G)I scenarios in future studies.

Physiologically Based Pharmacokinetic Modeling to Describe the CYP2D6 Activity Score-Dependent Metabolism of Paroxetine, Atomoxetine and Risperidone.

The cytochrome P450 2D6 (CYP2D6) genotype is the single most important determinant of CYP2D6 activity as well as interindividual and interpopulation variability in CYP2D6 activity. Here, the CYP2D6 activity score provides an established tool to categorize the large number of CYP2D6 alleles by activity and facilitates the process of genotype-to-phenotype translation. Compared to the broad traditional phenotype categories, the CYP2D6 activity score additionally serves as a superior scale of CYP2D6 activity due to its finer graduation. Physiologically based pharmacokinetic (PBPK) models have been successfully used to describe and predict the activity score-dependent metabolism of CYP2D6 substrates. This study aimed to describe CYP2D6 drug-gene interactions (DGIs) of important CYP2D6 substrates paroxetine, atomoxetine and risperidone by developing a substrate-independent approach to model their activity score-dependent metabolism. The models were developed in PK-Sim®, using a total of 57 plasma concentration-time profiles, and showed good performance, especially in DGI scenarios where 10/12, 5/5 and 7/7 of DGI AUClast ratios and 9/12, 5/5 and 7/7 of DGI Cmax ratios were within the prediction success limits. Finally, the models were used to predict their compound's exposure for different CYP2D6 activity scores during steady state. Here, predicted DGI AUCss ratios were 3.4, 13.6 and 2.0 (poor metabolizers; activity score = 0) and 0.2, 0.5 and 0.95 (ultrarapid metabolizers; activity score = 3) for paroxetine, atomoxetine and risperidone active moiety (risperidone + 9-hydroxyrisperidone), respectively.

Pharmacologic Targeting of MMP2/9 Decreases Peritoneal Metastasis Formation of Colorectal Cancer in a Human Ex Vivo Peritoneum Culture Model.

BACKGROUND: Matrix metalloproteinases (MMPs) play a crucial role in tumour initiation, progression, and metastasis, including peritoneal carcinosis (PC) formation. MMPs serve as biomarkers for tumour progression in colorectal cancer (CRC), and MMP overexpression is associated with advanced-stage metastasis and poor survival. However, the molecular mechanisms of PC from CRC remain largely unclear. METHODS: We investigated the role of MMPs during peritoneal colonisation by CRC cell lines in a human ex vivo peritoneum model and in patient-derived CRC and corresponding PC samples. MMP2 and MMP9 were inhibited using the small-molecule inhibitors batimastat and the specific MMP2/9 inhibitor III. RESULTS: MMP2 and MMP9 were strongly upregulated in patient-derived samples and following peritoneal colonisation by CRC cells in the ex vivo model. MMP inhibition with batimastat reduced colonisation of HT29 and Colo205 cells by 36% and 68%, respectively (p = 0.0073 and p = 0.0002), while MMP2/9 inhibitor III reduced colonisation by 50% and 41%, respectively (p = 0.0003 and p = 0.0051). Fibronectin cleavage was enhanced in patient-derived samples of PC and during peritoneal colonisation in the ex vivo model, and this was inhibited by MMP2/9 inhibition. CONCLUSION: MMPs were upregulated in patient-derived samples and during peritoneal attachment of CRC cell lines in our ex vivo model. MMP2/9 inhibition prevented fibronectin cleavage and peritoneal colonisation by CRC cells. MMP inhibitors might thus offer a potential treatment strategy for patients with PC.

(Z)-Endoxifen and Early Recurrence of Breast Cancer: An Explorative Analysis in a Prospective Brazilian Study.

Adherence to treatment and use of co-medication, but also molecular factors such as CYP2D6 genotype, affect tamoxifen metabolism, with consequences for early breast cancer prognosis. In a prospective study of 149 tamoxifen-treated early-stage breast cancer patients from Brazil followed up for 5 years, we investigated the association between the active tamoxifen metabolite (Z)-endoxifen at 3 months and event-free survival (EFS) adjusted for clinico-pathological factors. Twenty-five patients (16.8%) had recurred or died at a median follow-up of 52.3 months. When we applied a putative 15 nM threshold used in previous independent studies, (Z)-endoxifen levels below the threshold showed an association with shorter EFS in univariate analysis (p = 0.045) and after adjustment for stage (HR 2.52; 95% CI 1.13-5.65; p = 0.024). However, modeling of plasma concentrations with splines instead of dichotomization did not verify a significant association with EFS (univariate analysis: p = 0.158; adjusted for stage: p = 0.117). Hence, in our small exploratory study, the link between impaired tamoxifen metabolism and early breast cancer recurrence could not be unanimously demonstrated. This inconsistency justifies larger modeling studies backed up by mechanistic pharmacodynamic analyses to shed new light on this suspected association and the stipulation of an appropriate predictive (Z)-endoxifen threshold.

Triple Targeting of HER Receptors Overcomes Heregulin-mediated Resistance to EGFR Blockade in Colorectal Cancer.

Current treatment options for patients with advanced colorectal cancers include anti-EGFR/HER1 therapy with the blocking antibody cetuximab. Although a subset of patients with KRAS WT disease initially respond to the treatment, resistance develops in almost all cases. Relapse has been associated with the production of the ligand heregulin (HRG) and/or compensatory signaling involving the receptor tyrosine kinases HER2 and HER3. Here, we provide evidence that triple-HER receptor blockade based on a newly developed bispecific EGFR×HER3-targeting antibody (scDb-Fc) together with the HER2-blocking antibody trastuzumab effectively inhibited HRG-induced HER receptor phosphorylation, downstream signaling, proliferation, and stem cell expansion of DiFi and LIM1215 colorectal cancer cells. Comparative analyses revealed that the biological activity of scDb-Fc plus trastuzumab was sometimes even superior to that of the combination of the parental antibodies, with PI3K/Akt pathway inhibition correlating with improved therapeutic response and apoptosis induction as seen by single-cell analysis. Importantly, growth suppression by triple-HER targeting was recapitulated in primary KRAS WT patient-derived organoid cultures exposed to HRG. Collectively, our results provide strong support for a pan-HER receptor blocking approach to combat anti-EGFR therapy resistance of KRAS WT colorectal cancer tumors mediated by the upregulation of HRG and/or HER2/HER3 signaling.

Assessing human urinary clomiphene metabolites after consumption of eggs from clomiphene-treated laying hens using chromatographic-mass spectrometric approaches.

The anti-estrogen clomiphene is prohibited in sports at all times. Yet, adverse analytical findings (AAFs) have increased since 2011. This is possibly due to improved analytical sensitivity, but also contamination of food of animal origin needs to be taken into consideration as a potential source of drug exposure. For instance, studies with laying hens that received orally administered clomiphene have shown a significantly increased egg production rate but, as a consequence, eggs were found to incorporate residues of clomiphene. In order to evaluate if the consumption of clomiphene-contaminated eggs can cause an AAF of a doping control sample, eggs obtained from an animal administration study with clomiphene were consumed by human volunteers. Each volunteer ate two eggs, and urine samples were collected and analyzed using routine doping control procedures. Subsequently, additional volunteers received a microdosed clomiphene capsule to compare the excretion profiles. Maximum urinary concentrations of hydroxy-clomiphene (HC) between 80 and 300 pg mL-1 were detected following the consumption of clomiphene-containing eggs, which would constitute AAFs if observed in athletes' doping control samples. In order to support the differentiation of potential routes of drug exposure, a method was developed which allows for the chromatographic separation of (E)-3-, (Z)-3-, (E)-4-, and (Z)-4-HC using a derivatization step. By comparing the peak areas of these metabolites, characteristic relative distribution patterns were found that assist in identifying AAFs resulting from clomiphene ingested via contaminated eggs and, thus, enable to distinguish clomiphene intake via contaminated eggs from the intake of microdoses or therapeutic dosages, e.g. for doping purposes.

Subunits of BK channels promote breast cancer development and modulate responses to endocrine treatment in preclinical models.

BACKGROUND AND PURPOSE: Pore-forming α subunits of the voltage- and Ca2+ -activated K+ channel with large conductance (BKα) promote malignant phenotypes of breast tumour cells. Auxiliary subunits such as the leucine-rich repeat containing 26 (LRRC26) protein, also termed BKγ1, may be required to permit activation of BK currents at a depolarized resting membrane potential that frequently occur in non-excitable tumour cells. EXPERIMENTAL APPROACH: Anti-tumour effects of BKα loss were investigated in breast tumour-bearing MMTV-PyMT transgenic BKα knockout (KO) mice, primary MMTV-PyMT cell cultures, and in a syngeneic transplantation model of breast cancer derived from these cells. The therapeutic relevance of BK channels in the context of endocrine treatment was assessed in human breast cancer cell lines expressing either low (MCF-7) or high (MDA-MB-453) levels of BKα and BKγ1, as well as in BKα-negative MDA-MB-157. KEY RESULTS: BKα promoted breast cancer onset and overall survival in preclinical models. Conversely, lack of BKα and/or knockdown of BKγ1 attenuated proliferation of murine and human breast cancer cells in vitro. At low concentrations, tamoxifen and its major active metabolites stimulated proliferation of BKα/γ1-positive breast cancer cells, independent of the genomic signalling controlled by the oestrogen receptor. Finally, tamoxifen increased the relative survival time of BKα KO but not of wild-type tumour cell recipient mice. CONCLUSION AND IMPLICATIONS: Breast cancer initiation, progression, and tamoxifen sensitivity depend on functional BK channels thereby providing a rationale for the future exploration of the oncogenic actions of BK channels in clinical outcomes with anti-oestrogen therapy. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc.

Raman Imaging and Fluorescence Lifetime Imaging Microscopy for Diagnosis of Cancer State and Metabolic Monitoring.

Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.

A human ex vivo coculture model to investigate peritoneal metastasis and innovative treatment options.

OBJECTIVES: Peritoneal metastasis (PM) is commonly observed in patients with colorectal cancer (CRC). The outcome of these patients is poor, with an average survival of only six months without therapy, which requires a better understanding of PM biology and new treatment strategies. METHODS: We established and characterized a human ex vivo peritoneal model to investigate the mechanisms of peritoneal seeding and possible treatment options. For this, CRC cell lines and patient-derived tumor organoids were cultured together with human peritoneum to investigate the invasion of malignant cells and the effects of local chemotherapy. RESULTS: Fresh human peritoneum was cultured for up to three weeks in a stainless steel ring system, allowing for survival of all peritoneal structures. Peritoneal cell survival was documented by light microscopy and immunohistochemical staining. Further, immunohistological characterization of the tissue revealed CD3-positive T-lymphocytes and vimentin-positive fibroblasts within the peritoneum. In addition, extracellular matrix components (collagens, matrix metalloproteinases) were localized within the tissue. Coculture with CRC cell lines and patient-derived CRC organoids revealed that cancer cells grew on the peritoneum and migrated into the tissue. Coculture with CRC cells confirmed that hyperthermal treatment at 41 °C for 90 min significantly enhanced the intracellular entry of doxorubicin. Moreover, treatment with mitomycin C under hyperthermic conditions significantly reduced the amount of cancer cells within the peritoneum. CONCLUSIONS: This human ex vivo peritoneal model provides a stringent and clinically relevant platform for the investigation of PM and for further elucidation of possible treatment options.

Computational Treatment Simulations to Assess the Need for Personalized Tamoxifen Dosing in Breast Cancer Patients of Different Biogeographical Groups.

Tamoxifen is used worldwide to treat estrogen receptor-positive breast cancer. It is extensively metabolized, and minimum steady-state concentrations of its metabolite endoxifen (CSS,min ENDX) >5.97 ng/mL have been associated with favorable outcome. Endoxifen formation is mediated by the enzyme CYP2D6, and impaired CYP2D6 function has been associated with lower CSS,min ENDX. In the Women's Healthy Eating and Living (WHEL) study proposing the target concentration, 20% of patients showed subtarget CSS,min ENDX at tamoxifen standard dosing. CYP2D6 allele frequencies vary largely between populations, and as 87% of the patients in the WHEL study were White, little is known about the risk for subtarget CSS,min ENDX in other populations. Applying pharmacokinetic simulations, this study investigated the risk for subtarget CSS,min ENDX at tamoxifen standard dosing and the need for dose individualization in nine different biogeographical groups with distinct CYP2D6 allele frequencies. The high variability in CYP2D6 allele frequencies amongst the biogeographical groups resulted in an up to three-fold difference in the percentages of patients with subtarget CSS,min ENDX. Based on their CYP2D6 allele frequencies, East Asian breast cancer patients were identified as the population for which personalized, model-informed precision dosing would be most beneficial (28% of patients with subtarget CSS,min ENDX).

Simulation-Based Assessment of the Impact of Non-Adherence on Endoxifen Target Attainment in Different Tamoxifen Dosing Strategies.

Tamoxifen is widely used in breast cancer treatment and minimum steady-state concentrations of its active metabolite endoxifen (CSS,min ENDX) above 5.97 ng/mL have been associated with favourable disease outcome. Yet, about 20% of patients do not reach target CSS,min ENDX applying conventional tamoxifen dosing. Moreover, 4-75% of patients are non-adherent, resulting in worse disease outcomes. Assuming complete adherence, we previously showed model-informed precision dosing (MIPD) to be superior to conventional and CYP2D6-guided dosing in minimising the proportion of patients with subtarget CSS,min ENDX. Given the high non-adherence rate in long-term tamoxifen therapy, this study investigated the impact of non-adherence on CSS,min ENDX target attainment in different dosing strategies. We show that MIPD allows to account for the expected level of non-adherence (here: up to 2 missed doses/week): increasing the MIPD target threshold from 5.97 ng/mL to 9 ng/mL (the lowest reported CSS,min ENDX in CYP2D6 normal metabolisers) as a safeguard resulted in the lowest interindividual variability and proportion of patients with subtarget CSS,min ENDX even in non-adherent patients. This is a significant improvement to conventional and CYP2D6-guided dosing. Adding a fixed increment to the originally selected dose is not recommended, since it inflates interindividual variability.

Stereoselective quantification of phase 1 and 2 metabolites of clomiphene in human plasma and urine.

Clomiphene citrate is first line therapy of female infertility but is also frequently abused by athletes. Human biotransformation of clomiphene results in numerous phase 1 and phase 2 metabolites. The involvement of the polymorphic cytochrome P450 2D6 leads to a high inter-individual variability. To comprehensively investigate clomiphene metabolism in vivo we established a highly sensitive and specific UPLC-MS/MS method for the stereoselective quantification of clomiphene and its phase 1 and phase 2 metabolites in plasma and urine. Reference compounds and stable isotope labelled internal standards were synthesized in-house. High-throughput sample preparation was done by protein precipitation. Analytes were separated by UPLC on a C18 column (1.8 µm, 2.1 * 100 mm) using a gradient of 0.1% formic acid in acetonitrile in 0.1% aqueous formic acid and detected by positive ESI-MS/MS in MRM mode. The lower limit of quantification was below 1 nM for all analytes. The method was validated according to recent guidelines. However, due to absorption effects during sampling the quantification of metabolites in urine was limited to phase 2 metabolites. The method was successfully applied to determine the pharmacokinetic of (E)- and (Z)-clomiphene and 14 metabolites following a single dose of 100 mg clomiphene citrate in 3 healthy subjects and proofed to be an essential tool to comprehensively investigate the human biotransformation of clomiphene.

Metabolic Drug Response Phenotyping in Colorectal Cancer Organoids by LC-QTOF-MS.

As metabolic rewiring is crucial for cancer cell proliferation, metabolic phenotyping of patient-derived organoids is desirable to identify drug-induced changes and trace metabolic vulnerabilities of tumor subtypes. We established a novel protocol for metabolomic and lipidomic profiling of colorectal cancer organoids by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) facing the challenge of capturing metabolic information from a minimal sample amount (<500 cells/injection) in the presence of an extracellular matrix (ECM). The best procedure of the tested protocols included ultrasonic metabolite extraction with acetonitrile/methanol/water (2:2:1, v/v/v) without ECM removal. To eliminate ECM-derived background signals, we implemented a data filtering procedure based on the p-value and fold change cut-offs, which retained features with signal intensities >120% compared to matrix-derived signals present in blank samples. As a proof-of-concept, the method was applied to examine the early metabolic response of colorectal cancer organoids to 5-fluorouracil treatment. Statistical analysis revealed dose-dependent changes in the metabolic profiles of treated organoids including elevated levels of 2'-deoxyuridine, 2'-O-methylcytidine, inosine and 1-methyladenosine and depletion of 2'-deoxyadenosine and specific phospholipids. In accordance with the mechanism of action of 5-fluorouracil, changed metabolites are mainly involved in purine and pyrimidine metabolism. The novel protocol provides a first basis for the assessment of metabolic drug response phenotypes in 3D organoid models.